Diagnostic composition for isolating and identifying yeasts



United States Patent This invention relates to improvements in diagnostic compositions useful in isolating and identifying yeasts and to methods for employing such compositions for such purpose. The term yeasts as used herein does not imply any taxonomical classification but is employed in the sense of its general usage to designate the organisms with which the present invention is concerned.

In the past decade the increasing appearance of yeasts in pathological conditions has provoked a need for means to isolate, separate, and clearly identify them in order that those concerned therewith may approach their eradication, when desirable or necessary, with maximal certainty and assurance. Failure to recognize the nature of a yeast influencing a pathological condition may result in undue delay and misapplication of proper therapeutic techniques for the relief of such condition. This is, of course, undesirable.

The problem of yeasts, their isolation and identification has not gone unnoticed in the art. Several proposed means aimed at this problem have appeared and have met with some success. Notably, a composition developed by MacLarenet aL'and reported in the Americal Journal of Clinical Pathology, 30: 411-422 (1958) has been used in the art to isolate and distinguish various yeasts. However, this, and other means have not wholly solved the problem.

It is an object of this invention to provide a diagnostic composition having an enhanced capacity to differentiate among closely related yeasts involved in pathologic conditions.

It is another object of this invention to provide diagnostic compositions which are readily prepared and can be stored for long periods without substantial change.

It is still another object of this invention to provide diagnostic compositions which are adapted to be used readily in small tubes as well as the commonly employed petri plate, thus permitting greater facility in isolating and identifying yeasts.

It is a further object of this invention to provide diagnostic compositions adapted to readily discern the presence of mixed cultures.

Still another object of this invention is the provision of a diagnostic medium adapted to encourage the growth of yeasts while suppressing or inhibiting the growth of commonly encountered saprophytic or pathogenic bacteria whose growth tends to obscure and vitiate the characteristic growth and appearance of yeasts.

In accordance with the aforesaid objects the composition of this invention comprises a mixture of agar, carbohydrate, protein, surfactant, phosphomolybdic acid, an antibacterial agent and water. The agar employed in the preparation of this composition is the commonly used agar-agar derived from certain seaweeds and serves as the solidifying agent. The carbohydrate source is conveniently supplied by sucrose, while the protein requirements are met through the use of proteose-peptone (Difco) and yeast nitrogen base (Difco). The surfactant is preferably of the nonionic type as exemplified by the Tween series (Atlas Chemical Industries). The antibacterial agent may be chosen from those known to exert substantially no eifect upon yeast growth while effectively suppressing bacterial growth. Exemplary of such agents are neomycin, penicillin,

streptomycin and the like. The phosphomolybdic acid is that having substantially the formula (Merck). Water is, of course, the major ingredient, serving as a carrier.

The following description is exemplary of the preparation of the composition of this invention:

A. Stock medium Agar (1.5 gms.), sucrose (4.0 gms.) and proteose peptone (1.0 gm.) are added to water ml.) in a suitable vessel which is heated in a water bath until all ingredients are dissolved. The solution is autoclaved at 10 lbs. pressure for 15 minutes and then allowed to harden. It is then remelted in a water bath and then allowed to cool to a molten state (40-50) and held in this state awaiting the addition of other ingredients.

B. Solutions dissolving streptomycin sulfate and potassium penicillin G in' water so that each milliliter contains 2000 mcg. of streptomycin and 800 units of penicillin. In lieu of the penicillin-streptomycin solution, neomycin solution containing 10 mg. of neomycin in each milliliter may be used.

C. Diagnostic medium The diagnostic medium is prepared by combining 5 ml. of (l) with thorough shaking, 1 ml. of (2), 3 ml. of (3) and 5 ml. of (4) with the molten stock medium A.

The pH of the diagnostic medium thus prepared is about 5.2. It can be stored until needed by pouring in sealed petri plates or preferably in vials. A 2 dram vial containing about 3.5 ml. of diagnostic medium is preferred since it is readily stored and convenient to use. In storage there is no evidence of deterioration of this medium. Hence, it can be retained and obviates the necessity to make a fresh batch each time occasion arises for its use.

It is a feature in the preparation of this diagnostic composition that the incorporation of a surfactant abets the introduction of the phosphomolybdic acid into the stock medium A. and also suppresses the tendency to froth formation.

It has been found that when the diagnostic composition of this invention is contacted with various yeasts there is produced a characteristic colony coloration, characteristic colony growth and characteristic pigmentation of the medium. Thus, when yeasts of the genera Candida, Saccharomyces, T orulopsis and Tn'chosporon are exposed to this diagnostic composition at room temperature for a period of from 3-4 days the color of the colony ranges from white to olive to tan to gray to blue having gradation in shade as well as distinctive growth behavior and pigmenting characteristics. These functional diiferences in the presence of the diagnostic composition of this invention enable an identification of yeasts of pathologic concern.

In the table below is presented the behavior of various yeast species in the presence of the composition of this invention:

3 Yeast: Identifying characteristics Candida albicans An olive colony. Candida tropicalis A deep blue colony Candida albicans var.

stellatoidea T orulopsis glabrata A From the foregoing it is apparent that various yeast species react differently in contact with this diagnostic composition producing responses permitting difierentiation. Color production is enhanced due to the presence of the surfactant and bacterial growth suppressed to a noninterfering level by the presence of an antibacterial agent.

In carrying out the practice of this invention a sample obtained by a loop or sterile swab is implanted in the diagnostic composition which is maintained at room temperature (2025 C.) for a period of 34 days, after which the characteristics and appearance of the sample are observed and differentiated. In this way a positive identification of a single yeast organism or of a plurality of such organisms is secured in accordance with the teachings of this invention thus enabling appropriate and direct steps for dealing therewith to be taken.

What is claimed is:

1. A diagnostic composition useful in the rapid and positive identification of yeast species comprising:

Gms. Agar 1.5 Sucrose 4.0 Proteose peptone 1.0

dissolved in 100 ml. of Water and subjected to autoclaving, cooling, and remelting and having dispersed therein aqueous solutions consisting of (a) phosphomo-lybdic acid in the proportion of mgs./ 3 ml. H O, (b) polyoxyethylene sorbitan monolaurate in the proportion of 1 ml./ 8.75 ml. H O; (c) yeast nitrogen base in the proportion of 6.7 gm./ ml. H 0 and (d) an antibacterial agent in the proportion of 800 units of penicillin and 2000 mcg. of streptomycin/m1.

2. The composition of claim 1 wherein the antibacterial agent is neomycin in the proportion of 10 mg./ml.

References Cited UNITED STATES PATENTS 3/1957 Goldman -100 1/1961 Pagano et a1. 195l03.5

OTHER REFERENCES MacLaren et al.: American Journal of Clinical Pathology, vol. 3 0, pages 411-422 (1958).

ALVIN E. TANENHOLTZ, Primary Examiner.

R. R. JONES, Assistant Examiner. 

1. A DIAGNOSTIC COMPOSITION USEFUL IN THE RAPID AND POSITIVE IDENTIFICAION OF YEAST SPECIES COMPRISING: 